HIV(+) women have high risk of cervical precancer and cancer as well as infection with human papillomavirus (HPV), the viral cause of cervical precancer/cancer. Recent advances in genetic/epigenetic methods provide previously unachievable opportunities to study the HPV viral factors and other local infectious influences on the natural history of HPV and development of cervical disease in HIV(+) women. Under this proposal, we will use next generation (next-gen) sequencing to conduct precision HPV genomic analysis able to determine whether a given HPV type detected at two or more time points is the same exact viral infection versus different HPVs of the same type. These data will be used to comprehensively study: type-specific differences in HPV persistence and their relation with precancer; the occurrence of HPV reactivation and how often reactivated HPV persists and leads to precancer; the impact of immune status on each of these steps in HPV natural history. If cervical HPV can reactivate and progress to precancer, it would preclude screening cessation at age 65 years (which is done in the general population). Furthermore, HPV DNA methylation will be studied, as we found methylation in the HPV L1/L2 region very strongly associated with precancer/cancer, and pilot data suggest similar associations between methylation and precancer may exist for HIV(+) women. This research therefore will provide insight into the epigenetic modifications related to the development of cervical disease, and could possibly be used to improve the specificity and positive predictive value of HPV testing. Importantly, the cervicovaginal microbiome may influence both the HPV natural history and HPV DNA methylation and will also be studied. A pilot study by our group showed reduced HPV prevalence with high relative abundance of Lactobacillus crispatus but not other Lacobacillus species, and transition to a L. crispatus community state type was associated with reduced incident HPV. The L. crispatus results were especially promising since the protective effects were observed even with low CD4. However, the microbiota and HPV relationship needs to be studied in appropriately designed HIV(+) cohort studies of adequate size before any future probiotic intervention studies may be considered. There are currently little data regarding the impact of the microbiota on HPV natural history/progression (building on Aim 1). We will also study the microbiota and HPV methylation (building on Aim 2), as local microbiota was shown to alter methylation in neighboring tissue. Overall, these integrated studies address critical aspects of HPV natural history/progression, with significant implications to cancer prevention. This study will utilize semiannually collected specimens from the WIHS, the largest, long term cohort of HIV(+) (N=2793) and high risk HIV(-) (N=975) women. The Specific Aims are, in HIV(+) women, to study: (i) The role of reactivation in HPV natural history and precancer risk, using next-gen ?precision? HPV genomic assays; (ii) HPV DNA methylation and its relation with precancer risk; The microbiome's impact on HPV natural history, HPV methylation, and HPV progression.